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238 PART III. ABNORMAL HEMOGLOBINS 4. Ingram, V. M.: A specific chemical difference between the globins of normal human and sickle-cell anaemia haemoglobin, Nature 178: 792, 1956. 5. Schroeder, W. A., Rhinesmith, H. S., and Pauling, L.: In press. 6. Stein, W. H.: These Proceedings and in press. 7. Neel, J. V.: Inheritance of sickle cell anemia, Science 110: 64, 1949. 8. Benzer, S.: The elementary units of heredity, in The Chemical Basis of Heredity, McElroy, W. D., and Glass, B., eds., Johns Hopkins Press, Baltimore, 1957. 9. Streisinger, G., and Franklin, N. C.: Mutation and recombination at the host range genetic region of phage T2, Cold Spring Harbor Symp. Quant. Biol. At: 103, 1956. 10. Pritchard, R. H.: The linear arrangement of a series of alleles of ~spergillas Nidula7~s, Heredity 9: 343, 1955. 1 1. Giles, N. H., Partridge, C. W. H., and Nelson, N. J.: The genetic control of adenylosuccinase in Neurospora (grassy, Proc. U. S. Nat. Acad. Sci. 43: 305, 1957. 12. Itano. H. A.: The hemoglobins, Ann. Rev. Biochem. 25: 331, 1956. 13. Zuelzer, W. W., Neel, J. V., and Robinson, A. R.: Abnormal hemoglobins, Prog- ress in Hematology 1: 91, 1956. DISCUSSION Dr. falter L. Hughes: After Dr. Ingram's very beautiful sleuthing job, I hate to get down to a very practical problem, but I do avant to initiate a discussion on sulfhydryl groups in hemoglobins. All my analyses have been done by methyl mercury determinations. We have used methyl mercury as the most specific reagent known to me for -SH interaction. Certainly everyone agrees that mercury forms the most specific bonds with sulfur, and methyl mercury, the smallest mercurial, should react most easily. To initiate the discussion, I will briefly present the data I have accumulated sporadically over the years. I have studied three species of hemoglobinbovine, horse, and human and found -SlI contents for bovine, 2; horse, 4; and~human, 6. More par- ticularly, I have found two such groups in bovine hemoglobin, whether native or denatured, and also in globin isolated from this hemoglobin. In the case of equine hemoglobin, whether native or denatured, I have found four groups. I might say that these values are not exact integers. However, I believe the sulfhydryls must be an integral part of the molecule. I do not think Dr. Stein's finding of impurities can explain all of these species differences and so I have rounded off the values. The precise values obtained were: for bovine, 2 to 2.5; for equine, 4 to 4.5; and for human, 5.5 to 6. The bovine hemoglobin was not crystallized and I suspect some impurity may be raising the value obtained. The equine and human have been crystal- lized. In the case of the human hemoglobin, there appear to be two types of sulfhydryl groups; two are very active and four more are less active and can hardly be recognized completely except on denaturation. I think I can draw from published data arguments in support of my own.

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DISCUSSION 239 Dr. Ingram is here and so I do not mind presenting his data, even though I have taken some liberties with them. These are his data of two years ago.i (Table I). He has prepared a more recent paper which I have not seen and upon which he may wish to comment. TABLE I -SH CONTENT OF HEMOGLOBINS Reagent AgN O3 HgCl~ HgCl., PCMB Moles Bound Bovine Human 4.0 8.3 6 2 Moles of Mercurial Abolishing Silver Binding (2) 6 3 -a} * Adapted from: Ingram, V. M., Sulphydryl groups in haemoglobins, Bioch. J. so: 6D3, 1955. If you compare the bovine and human hemoglobins which Dr. Ingram studied, you will notice that he indicates that bovine has four sulfhydryl groups by silver titration, but he finds that two mercuries will block all four. Simi- larly, he finds eight in human hemoglobin by silver titration which can be blocked by six mercuries. He obtains a similar picture with mercurials. Since the more specific mercurial can completely block the excess silvers, I believe this rules out the possibility that the extra groups can be sulfhydryl. They must instead be some other group which is titrating with an amenity for silver close enough to that of suliLydryl so that they are included in the curvature at the end point of the amperometric titration. I think the final word on the -SH content will come via some good sulfur and methionine de- terminations which will establish an upper limit to the sulihydryl values. I can cite what I believe are the best data on equine hemoglobin. Trickery re- ports eight sulfurs and Brand3 reports four methionines. Then by difference we obtain a maximum of four -SH groups in perfect agreement with our sulfhydryl analysis. REFERENCES 1. Ingram, V. M.: Sulphydryl groups in haemoglobins, Biochem. J. 59: 653, 1955. 2. Trickery, H. B., and White, A.: Proportion of cystine yielded by hemoglobins of the horse, dog and sheep, Proc. Soc. Exper. Biol. and Med. 31: 6, 1933. 3. Brand, E., and Grantham, J.: The methionine and isoleucine content of hemo- globins, J. Am. Chem. Soc. 63: 724, 1946. Dr. R. Benesch: I would like to mention an experiment which confirms the very interesting suggestion first made by Dr. Ingram that title -SH groups of hemoglobin occur in closely-spaced pairs or ever triplets. It is possible to dis- place the proton of a sulfhydryl group quantitatively with ~ specific reagent (Benesch, R., and Benesch, R. E.: Biochim. Biophys. Acta 23: 643, 1957~.

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240 PART III. ABNORMAL HEMOGLOBINS Since there are four reactive -SH groups in native human hemoglobin, the liberation of four protons would be expected upon treatment with a protorl- displacing reagent, such as salyrgan in the RlIgCl form (4 RHgC1 + Pr (SH ) 4 Pr ( SHgR) 4 + 4H+ + 4 Cl- ) . The value which we found, however, was two, thus confirming the contention that reaction of one -SH group of a pair with this large mecurial blocks the other one to access by the same reagent. A final point which has general applicability in protein chemistry. Many proteins, including hemoglobin, have been treated with urea in order to de- nature them and to break hydrogen bonds. What is not often realized is that urea is capable of forming quite stable complexes with metals, iron being one of them. Barbieri (Barbieri, G. A.: Atti accad. naz. Lincei, Vol. 22 No. I 867, 1913) was the first to crystallize hexa-(urea)-iron (III) chloride, o4 0-3 O D oo t - O xx t ~ 30 mans ~ t = 20 hrs 02 01 _ /! / i/~\ ~ ~ o ' ' ' ~ ~ _ 520 540 560 580 600 ~ (mix) FIG. 1. Denaturation of hemoglobin in the presence of urea and silver ions. TABLE I -SH GROUPS AND STABILITY OF HEMCGLOBIN Tris buffer pH 7.4 To decrease time in ~ 576 Tris buffer pH 7.4 Urea 4 M To decrease time in ~ 576 Human Hb control 20 furs. 2 20 furs. 5.5 8 moles Ag+ per mole protein 30 mins. 41 same 75 mins. 3.6 75 " 49 same 270 " j 1 same 20 furs. 14 20 furs. 59 Sheep Hb control 20 furs. 14 20 furs. 14 8 moles Agm per mole protein 20 " 21 20 " 29

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DISCUSSION 241 (Fe(OC(NH~6)Cl3. This is one fact which should be taken into account when one treats a protein like hemoglobin with urea. This is illustrated in the figure and the table. The findings may be sum- marized as follows: When human oxyhemoglobin is treated with eight atoms of silver to block what we believe are all the sulfhydryl groups, nothing much happens to the hemoglobin. Likewise, nothing happens to the hemoglobin when it is treated with urea. When the protein is treated with eight atoms of silver in 4 ~ urea, however, a very rapid breakdown takes place (fig. 1 and table I). As illustrated here, the breakdown is essentially to me/hemoglobin, although it then proceeds further. In summary, what it really comes to is that when the -SH groups are blocked, the urea is capable of pulling the iron out of the molecule. Dr. Howard Dintzis: In support of Dr. Hughes' observation and in th matter of chemical semantics, I should like to point out that the people who are titrating with silver and mercury are not necessarily titrating -SH groups. This is a matter of great confusion at present. They are titrating silver and mer- cury binding sites. Evidence from x-ray crystallography on hemoglobins cer- tainly is that, under some conditions, there are groups other than the -SH groups which combine mercury. We showed this by blocking the -SH groups and found that mercury was still bound under some conditions. In myoglobin the situation is very similar. There are groups that bind both mercury and silver even though there is no -SH group under some conditions. It was found that there are specific binding sites for silver and specific binding sites for some mercurials. This point should be made clearer than it has been so far. Dr. Max Perutz: There is some x-ray evidence regarding the location of the -SH groups in hemoglobin. You have seen on the diagrams which I showed yesterday that there are two -SH sites in hemoglobin which can be blocked by mercury. The same two sites are found if hemoglobin is blocked with four silver atoms making fIbAg4. The second pair of silver atoms goes close to the first pair, the four silver atoms giving rise to only two oblong peaks on a Fourier projection, each peak apparently representing two silver atoms. This kind of picture has been obtained both in horse and bovine hemo- globin, which are the only two species so far examined. I do not know what the picture would look like in human and in sheep hemoglobin, but I should like to express the hope that in due course we shall all agree on all these hemoglobins having just four -SH groups and no more. Dr. V. M. Ingram: In discussions with various people who have used these methods it has become quite clear that, under truly identical conditions and with samples prepared in the same way, there is very good agreement on the numbers of silver or mercury atoms which are bound by a protein. Disagree- ments have arisen because we were not aware of the profound effect of altera- tions in the experimental conditions. I was very glad to hear Dr. Dintzis' remarks. I, too, think that we should

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242 PART III. ABNORMAL HEMOGLOBINS talk of silver and mercury binding sites and not assume that those will neces- sarily be -SH groups. No doubt most of them will be sulihydryl groups, but, as the work of Dr. Stein and his colleagues has shown, independent con- ~ . . Creation Is necessary. In the light of his experiments it would appear that there are no more than six -SH groups in normal human hemoglobin. My earlier findings that eight silver atoms and six mercury atoms are bound by the denatured protein seem to need a new explanation. One could assume that the six -SH groups bind si:; silver or mercury atoms and that, in addition, two of them are capable of binding an additional silver atom each. Such a compound has been postulated by Cecil. Whereas the original interpretation called for two pairs of Ag atoms held by two pairs of very closely spaced -SH groups, we now have two very close pairs of Ag atoms held by only two individual sulfhydryl groups. The mercury binding on sites which cannot be -SH groups, which Dr. ~~ntz~s mentioned, is very much less firm and of quite a different order of magnitude. It is not detectable under the conditions of amperometric titration. As far as Dr. Hughes' contribution is concerned, I am glad that we seem to be in complete agreement on mercury binding in denatured hemoglobins. Two, four and six mercuries are the numbers which I, too, found in denatured ~ . . . . . Ox, horse and human hemoglobin. In addition, we also agree that in human hemoglobin two mercuries are bound very readily and the other four not so firmly; in fact, I found that one has to denature the molecule before they will combine. Dr. [MY. H. Stein: In a protein such as hemoglobin where the absolute values for -SH groups or half-cystines are low, it is difficult to be certain of the results to better than +1 residue per molecule. Hence, the preserve of four, five, or six -SH groups per molecule would be compatible with our data. Eight residues seems unlikely. We would certainly agree with the previous speakers that amperometric titrations can, under various conditions, yield differing results. It is for this reason that the determinations of half-cystine as cysteic acid were performed. The cysteic-acid method has been checked in a number of different ways in the past, and though not necessarily foolproof, does not possess the same kind of uncertainties found in the titration pro- cedures. Moreover, it is difficult to see how hemoglobin can contain more -SH groups by amperometric titration than there are half-cystine residues. A higher value by amperometric titration than by the cysteic-acid method would appear to favor the presence of binding sites for silver or mercury other than -SH groups, as has been mentioned by others. Such a discrepancy was not encountered in our studies, however. I should like to return to Dr. Ingram's very beautiful work for a moment, because it seems to me to carry an important message relative to the homo- geneity of hemoglobin. If hemoglobin were a complex mixture of proteins, treatment with trypsin would be expected to yield a far greater number of

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DISCUSSION 243 peptides than would be predicted from the arginine plus lysine content, assuming the protein to be pure, and the yields of these peptides would be low. I gather, however, that Dr. Ingram is getting about the expected number of peptides, and that the yields are high. A similar kind of result was obtained by Sanger with insulin, and in our own studies on ribonuclease, and these two proteins are of quite well-established homogeneity. Dr. Ingram's work would thus impose quite definite limits on the heterogeneity-of the hemoglobin he employed, and would indicate that the source of the gross heterogeneity found by other procedures must reside in structural differences which are not re- flected in alterations in the sequences of the amino acid residues. Would you agree to that, Dr. Ingram? Dr. Ingram: Yes, I would. Dr. John F. Taylor: I think it is important to point out that we may some- times be interested in the total -SH and sometimes in the number of -SH groups that are detectable in native hemoglobin under mild conditions. The former is evidently related to the total hemoglobin sulfur and its partition, about which there is still some uncertainty. The latter appears to be the more significant in relation to hypotheses about the mechanism of heme-heme in- teractions or other aspects of hemoglobin structure and function. We have measured the apparent -SH of several native mammalian hemo- globins,~ with the different reagents that have beer discussed, including 1 .1 1 1 I /1 ~ ~X7-,L ~ _ , _ ~ , ~ silver, methyl mercury and t~iV1~. W 1tn any one hemoglobin preparation the extent of reaction depends upon the reagent and, especially with silverer, upon the reaction conditions. Inasmuch as PC~B treatment affects the heme- heme interactions, the number of -SH sites revealed with this reagent seems especially significant. With any one reagent the number of apparent -SH groups depends upon the source of the hemoglobin and its history. Careful handling and the use of EDTA in the preparation of a sample favor the preservation of its titratable -SlI groups. Inasmuch as heme-heme interactions are rather similar among the mammalian hemoglobins, while the number and the nature of the PC~B-reactive sites differ, the more significant number of sites would seem to be the smallest that has thus far been observed. This number is two in bovine hemoglobin and also, with PLUMB, in human hemo- ~l~hin Fach site might include more than one -SH group, of course. There are several indications that not all the reactive -SH groups are identical, when more than two are detectable. For example, in canine hemo- globin, which reacts with four moles of PCNIB (per Hb of 68,000) taco sites appear to differ from the other two, both in their reaction with ferricyanide (described on page 155) and in their reaction with cystine. Two of the four PCMB-reactive sites of canine oxyhemoglobin, and both of the two sites of bovine and human oxyhemoglobin, are abolished upon treatment with excess cystine at pH 9. From the resulting increase in electrophoretic mobility, com- pared with untreated HbO~, on either side of the isoelectric pH, it may be a, _ e,

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244 PART III. ABNORMAL HEMOGLOBINS suggested that cystine reacts with the hemoglobin -SH to form a mixed disulfide: HEMO-~-SlI + 2 C S SC ~ HE)/IO-~-S-SCy Eldjarn and Pihl' have described and studied quantitatively the analogous re- action of cystamine with several proteins, including hemoglobin. The reaction j ust described, applied to several mammalian hemoglobins which contain differing numbers of reactive -SH groups in the native state, leas enabled us to prepare a series of hemoglobins that have been modified with respect to their titratable -SH groups without detectable change in absorption spectrum or in their ability to form oxy- or me/hemoglobin. Some samples of modified canine hemoglobin have been crystallized. The properties and re- actions of these substances are under investigation. Depending upon the sub- stance used to react with the hemoglobin, it will also be possible to vary the number and kind of the polar or non-polar side chains introduced into the protein in place of -SH. Finally, by extending the methods developed by Eldjarn and Pihl it will be possible to use the above reaction as another inde- pendent measure of the number of reactive -S. H sites in the molecule of hemoglobin or other protein. + 2CySH . REFEREN CES 1. Taylor, J. Fit.: Sulfhydryl groups of hemoglobin, Third International Congress of Biochemistry, Resume of communications, p. 17, 1955. 2. Eldjarn, L., and Pihl, A.: On the mode of action of x-ray protective agents. I. The fixation in ciao of cystamine and cysteamine to proteins, J. Biol. Chem. 223: 341-352, 1956. Dr. R. Benesch: I agree, of course, that silver and mercury are bound by many groups besides -SH groups. However, the essential difference is that the affinity of these metals for -SH groups exceeds (usually by several orders of magnitude) that for other sites on the protein. Therefore, when proteins are treated with these reagents the -SH groups react first and only after these sites are saturated does further nonspecific interaction with other groups take place. This is illustrated by the equilibrium dialysis experiment with hemo- globin taken from the paper by Benesch, et. al., (Benesch, R. E., Lardy, H. A., and Benesch, R.: J. Biol. Chem. 216: 663, 1955.) (Fig. 1.) In the case of the amperometric silver titration, the specificity is further enhanced by the following factors: Owing to the high sensitivity of the rotating platinum electrode, the absolute concentration of protein, and therefore of silver, is extremely low (about 2 x 10-;'M>. This may be contrasted, for example, with the equilibrium dialysis against p-mercuribenzoate, shown below, where con- centration ten times higher had to be employed to obtain significant results .

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DI SCUSSION C 12 - o Q O 8 c o D US o E 245 fix x / O x ~ 4 x~ l/ / -/x~o x ~ 0 ~0 x O ok/ 0/ 0 / 0 - x-x = s he e p H b 02 0-0= human HbO2 , ~ I 10 20 30 p-Mercuri benzocte c x ~ 0 4 FIG. 1.Reaction of hemoglobins with p-mercuribenzoate. (From the Journal of Biological Chemistry 216: 672, 1955; by permission. ) 2. Only a comparatively small excess of silver is necessary for an accurate determination of the end point. 3. The silver is used in the form of a relatively stable complex arid will therefore have little tendency per se to react with groups other than -SH groups. 4. A long list of biologically occurring compounds, such as amino acids, nucleic acids, etc., has been found not to react with silver at all under the conditions employed in this method Dr. Makio M~crayama': I would like to carry this discussion further by re- porting, in some detail, our studies of titratable sulfhydryl groups of normal, sickle cell, "C" and fetal hemoglobins. It was observed in our laboratory* that a deoxygenated sickle cell hemo- lysate has a negative temperature coefficient of "elation, i. e., a deoxygenated , ~ sickle cell hemoglobin solution of sufficient concentration gels at 38 C.,~ but the gel liquifes upon being cooled to 0 C. The reaction is reversible. This observation suggested that some alteration ir1 the molecular architecture of hemoglobin occurs during the process of deaggregation, i. e., the complemerl- tary combining sites on adjacent hemoglobin molecules must be altered at 0 C. One of the objects of the present investigation was to learn whether there would be a change in the number of titratable -SH Groups between the ice point and body temperature. ~ 1 Riggs reported that a sickle cell hemolysate which gels at 38 C. loses its gelling property where dialyzed against deionized water. Oxygenation also prevents gel formation of a sickle cell hemolysate.7 s, ii We investigated the relative accessibility of -SH groups at 0 and 38 C. for the following prep- arations: a) normal alla sickle cell deoxygenated hemolysates; b) normal and sickle cell oxygenated hemolysates; c) normal and sickle cell hemoglobins ~ While Dr. A. C. Allison was visiting the Gates and Crellin Laboratories of Chemistry, California Institute of Technology, in the summer of 1954.

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246 PART III. ABNORMAL HEMOGLOBINS crystallized and then dialyzed against distilled water, deoxygenated; d ) normal and sickle cell hemoglobin solution crystallized and then dialyzed against distilled water, oxygenated; and e) hemoglobin C and normal fetal hemoglobin crystallized and then dialyzed against water, deoxygenated. I will summarize the data obtained by argentometric-amperometric titra- tior~ methods, as well as the mercurimetric-amperometric titration technique on Hb A and Hb So ~ and Hb C and Hb F.4 ~ It was found that the maxi- mal number of titratable -SH groups is the same for Hb A, lIb S and Hb C; however, the mercurimetric-amperometric titration data suggest that their spatial arrangement appears to be different. The fetal hemoglobin molecule has fewer titratable -SH groups than Hb A and the mercapto group spatial arrangement is also different from that of the normal adult hemoglobin mole- cule. The hemolysates were prepared as has been described elsewhere.5 The apparatus t and technique used for amperometric titrations are essentially those described in the same reference.5 Resells and Conclusions. About four -SH groups per molecule are titra- table argentometrically for both normal arid sickle cell hemolysates at 0 C. There is one less titratable -SH group per molecule of normal hemolysate at 38 than at 0 C. There are two less titratable -SH groups per molecule of sickle cell hemolysate at 38 than at 0 C. In contrast, the number of mole- cules of PC~B bound by normal and sickle cell oxygenated hemolysates was essentially the same at 0 and 38 C. For undialyzed fresh hemolysates, about three moles of PCMB per oxyhemoglobin molecule are bound in both ir~- stances. A preliminary study of Hb C and Hb ~ indicates that there are about two to three PCMB-bindir~g sites in these molecules also. At O about four -SH groups per molecule are titratable for dialyzed ~ deoxygenated and oxygenated ~ hemoglobins A, S and C by the argento- metric method, whereas fetal (crystallized and dialyzed) hemoglobin binds about six silver atoms per molecule at both 0 and 38 C. The increment of titratable -SH groups equals plus four for hemoglobins A, S and C when the temperature of Tris buffer is raised from 0 to 38 C. The results of mercurimetric-amperometric titration were quite unexpected. About four mercury atoms are bound per molecule of normal and C hemo- g;lobins at 0; the result is represented very schematically in figure 1A. Since there are also about four argentometrically-titratable -SH groups at 0 C. in Hb A and Hb C molecules, it is suggested that the titratable -SH groups at 0 C. are located far apartthe -SH groups are separated so that no pair- ing is possible; thus, they form -S-Hg-C1 instead of -S-Hg-S-. On the other ~ An automatic amperometric titration apparatus is available; however, it was not used for this investigation. It is described in Encyclopedia of Instrumentation for Industrial Hygiene, Gaffe, C. D., Byers, K. H., and Mosey, A. D., eds., University of Michigan, Ann Arbor, 1956. See pp. 430~32, Automatic amperometric titration assembly.

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DISCUSSION 247 hand, dialyzed sickle cell hemoglobin binds about three mercury atoms per molecule at 0; this is represented schematically in figure 1B. It is suggested that one mercury atom could be bound ~ in -S -Hg-S-, the centers of the sulfur atoms are separated by twice the covalent Hg S bond distance, giving -S-!Ig-S 5.60 A) and that the other two titratable -SH groups are sepa- rated too far. A B O O . . O O O 0~0 0 , o 5.60A FIG. IA. A schematic representation . . . Of the spatial arrangement of titratab~e -SH groups at 0 of dialyzed normal hu- mar~ hemoglobin molecule as well as for Hb-C molecule. FIG. 1B. A schematic representation of the spatial arrangement of titratable -SH groups at 0 of dialyzed sickle cell hemo- globin molecule. A O O B O O O O . O O ~ A,..... O 5.60A O O O O O ,... O O - .~ O >5.60A O O O ~ . O O -..._: O 5.60A O O O ~ ~ . ~ o 5.60A O O ~ --~ O O C D FIG. 2A. -A conformation of sulfur atoms (of titratable -Sf1 groups at 38 ) of normal adult hemoglobin molecule. FIG. 2B. A conformation of sulfur atoms (of titratable -SKI groups at 3g ) of sickle cell hemoglobin molecule. FIG. 2C. A conformation of sulfur atoms (of titratable -SH groups at 38) of the "C" hemoglobin molecule. FIG. 2D. A conformation of sulfur atoms (of titratable -SH groups at 0-and 38) of the normal fetal hemoglobin molecule. (Figure 2AD appears in Federation Pro- ceedings 16: 758, 1957, and is reproduced by permission of the publishers.) At 38 C. about six mercury atoms are bound per molecule of dialyzed normal deoxygenated hemoglobin. There are about two mercury atoms more than at 0. In the argentometric titration an increase of about four Ag atoms per molecule is obtained when going from 0 to 38 C. It is suggested that the four -SH groups which become titratable ~ accessible ~ at 38 are so arranged that two mercury atoms can be bound by them. This is schematical- ly shown in figure 2A. It can be seen that two -S-Hg-S- linkages are possible in this molecule. Dialyzed sickle cell hemoglobin binds about five mercury atoms per mole-

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24S PART III. ABNORMAL HEMOGLOBINS cute at 38. This is about two atoms of mercury more than at 0. For silver the increase leas about four atoms per molecule. In accordance with the previous picture, this could mean that four new -SH groups are close enough (two and two) to be handled by just two mercury atoms. This is schemat- ically shown in figure 2B. It can be seen that sickle cell hemoglobin probably has three -S-Hg-S- linkages at the equivalence point in the mercurimetric- amperometric titration. Dialyzed hemoglobin C binds about eight silver or eight mercury atoms per molecule at 38. Furthermore, the data suggest that eight titratable -SH groups are separated far apart these are all "lone" -SH groups; at the equivalence point of the mercurimetric-amperometric titration eight -S-Hg-Cl's are formed. These findings are schematically represented in figure 2C. As can be seen from the schematic diagram, there are no -S-Hg-S- bridges pos- sible (centers of the sulfur atoms are separated by a distance greater than S.60 A) in the Hb C molecule. The total number of titratable -SH groups is the same for Hb A, fIb S. and Hb C, but their spatial arrangement appears to be different. The amino acid compositions of these hemoglobins are similar.)' -! i~ Pauling et al.~, suggested that the difference in structure between a "normal" and an "ab- normal" hemoglobin molecule may be a difference in the way in which the polvpeptide chains are folded. The present findings suggest that there is one . . T ~ ~ . T ~ ~ A ~ 1 mercapto pair more in rib ~ than in lib ~ and, furthermore, that there are two mercapto pairs less in Hb C than in Hb A. There are six titratable -SH groups in fetal hemoglobin "purified" from the cord blood. Dialyzed Hb F binds about six silver and about three mer- cury atoms per molecule at 0 as well as at 38. There are three -S-Hg-S- linkages possible in this molecule, which is schematically represented in fig- ure 2D. Discussion. Pauling's steric hindrance theory of heme-heme interaction in hemoglobin provides an obvious explanation of the action of oxygen in pre- venting the sickling of sickle cell anemia erythrocytes as well as the gelling of the sickle cell hemolysates.8 He has visualized the sickling process as one in which complementary sites on adj acent hemoglobin molecules combine. It was suggested that oxyhemoglobin and carbonmonoxyhemoglobin do not aggregate because of steric hindrance of the attached oxygen or carbon monoxide molecules. This steric hindrance effect might distort the comple- mentary sites through the forcing apart of layers of protein, as suggested by the isocyanide experiments. It was known for a long time that oxygen exerts a marked inhibitory effect ore the sickling produced by any means. Thus oxygen also exerts an inhibitory effect on the sickling produced by sulfhydryl compounds and is capable of reversing the phenomenon. Thomas and Stetsoni4 reported that sulihydryl compounds, notably FINS, 2,3-dimercaptopropanol (BAL), and cysteine,

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DISCUSSION 249 Fuse sickling. They also found that sickling is completely inhibited by mall amounts of -STI blocking agents (o-chloromercuribenzoate, iodoaceta- .~ide, iodosobenzoate, sodium maleate ~ . Ions of various heavy metals also inhibit sickling. These -SH blocking agents presumably act to destroy the complementariness of configuration of the surface ot the molecule. vv nen deoxygenated sickle cell hemoglobin gel (at 38) is cooled to 0, the com- plementary sites must be likewise altered, resulting in liquefaction of the gel. An architectural alteration of the sickle cell hemoglobin molecule appears to be reflected by the change in the number of titratable -SH groups. Hb A and Hb C also undergo architectural alterations faith temperature, whereas Rib F does riot. ~ ~ 1 1 1 ~ to Not all of the -STI groups of normal and sickle cell hemoglobin molecules are titratable in the hemolysates. The -SH groups are more accessible to the titrants (Ag~ and Hgtt) after dialysis against water. There are four -SH groups per molecule not titratable at 0 in these hemoglobins. These four non- titratable groups might be imbedded in the protein moiety; they are not titra- table probably due to steric hindrance between -SH groups and a part of globin in the hemoglobin molecule. Similarly PC\1B binding is not equiva- lent to silver. About three PC~:B molecules are bound by oxygenated normal ar,d sickle cell hemolysates. The data suggest that there are about three -SH groups which are different: they are less sterically hindered by a part of the globin molecule. Dialysis of hemoglobin solution against water is known to produce con- , ~ ~ siderable alteration of the molecular architecture. For example, the oxygen ~ l 1 1-2 ~ - l _ - _ _ r L _ ~1 _ L 2 ~ affinity is increased many Iola.~> LJlalysls OI nemog;looln agalIl5t WE1~E 1b believed to cause loosening of the structure through the operation of electro- static repulsive forces between the similarly charged portions of the molecule. In the presence of salt, these repulsive forces are diminished by the ion at- ~nosphere that surrounds the charged groups. The loosening of the protein structure would make imbedded -SH groups of hemoglobin molecule easily accessible. Some of the -SH groups appear to be arranged in pairs, and they are able to bind mercury atoms forming -S-Hg-S- bridges. Evidence for the pairing of -SH groups comes from x-ray diffraction data. Studies of Perutz~ and Perutz, Liquori' and Eirichi have shown that the molecules of horse and human hemoglobin possess dyed axis of symmetry. Furthermore, -ray dif- fraction measurements of horse hemoglobin with four equivalents of silver showed only one slightly elongated peak in the electron density map for each pair of silver atoms, indicating directly that two -SH groups must be close together. Acknowledgments: I wish to express my indebtedness to Prof. Linus Paul- ing for helpful suggestions and to Prof. Dan H. Campbell for his interest and encouragement during the investigation. I am also indebted to Prof. David

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250 PART III. ABNORMAL HEMOGLOBINS L. DraLkin for his interest in this problem. I thank Drs. Phillip Sturgeon and W. R. Bergren of the Children's Hospital in Los Angeles for providing blood specimens from patients with homozygous sickle cell anemia. I am also in- debted to Drs. Neva Abelson, l. D. Alexander, Carl E. Bechman, P. I. Jensen, A. McClements, and E. ~I:ertens, all of Philadelphia, who generously provided blood specimens. REFEREN CES 1. Harris, J. W.: Studies on the destruction of red blood cells. VIII. Molecular orientation in sickle cell hemoglobin solutions, Proc. Soc. Exper. Biol. & Med. 75: 197, 1950. 2. Huisman, T. H. J., Jonxis, l. H. P. and van der Schaaf, P. C.: Amino acid com- position of four different kinds of human hemoglobins, Nature 175: 902, 1955. 3. Murayama, M.: Titratable sulfhydryl grroups of normal adult and sickle-cell hemoglobins, Fed. Proc. 15: 318, 1956. T. Murayama, M.: Titratable sulfhydryl groups of fetal and "C" hemoglobins, Fed. Proc. 16: 223, 1957. 5. Murayama, M.: Titratable sulihydryl groups of normal and sickle-cell hemo- globins at 0 and 38, J. Biol. Chem. 228: 231-240, 1957. 6. Murayama, M.: (In preparation). 7. Pauling, L.: The hemoglobin molecule in health and disease, Proc. Am. Phil. Soc. 96: 556, 1952. 8. Pauling, L., Itano, H. A., Singer, S. J., and Wells, I. C.: Sickle cell anemia, a molecular disease, Science, 110: 543, 1949. 9. Perutz, M. F.: X-ray analysis of haemog;lobin, Nature 149: 491, 1942. 10. Perutz, M. F~., Liquori, A. M., and Eirich, F`.: X-ray and solubility studies of the haemoglobin of sickle-cell anaemia patients, Nature 167: 929, 1951. St. George, R. C. C., and Pauling, L.: The combining power of hemoglobin for alkyl isocyanides, and the nature of the heme-heme interactions in hemoglobin, Science 114: 629, 1951. 12. Schroeder, W. A., Kay, L. M., and Wells, I. C.: Amino acid composition of hemo- globins of normal negroes and sickle cell anemics, J. Biol. Chem. 187: 221, 1950. 13. Sidwell, A. E., Jr., Munch, R. II., Barron, E. S. G., and Hogness, T. R.: The salt effect on the hemoglobin-oxygen equilibrium, l. Biol. Chem. 121: 335, 1938. 14. Thomas, L., and Stetson, C. A., fir.: Sulihydryl compounds and the sickling phenomenon; a preliminary report, Bull. Johns Hopkins Hosp. 81: 176, 1948. 15. van der Schaaf, P. C., and Huisman, T. H. J.: The amino acid composition of human adult and foetal carbonmonoxyhaemoglobin estimated by ion exchange chromatography, Biochim. Biophys. Acta 17: 81, 1955. Dr. T. H. J. H2'isman (CommunicafionJ:* In recent years many studies have been carried out in order to increase our knowledge of the structure of the human hemoglobins. Especially in regard to the structure of fetal hemo- globin, the chemical analyses of the N-terminal residues, C-terminal residues arid sulfLydryl content seem of importance. It was found ~ ' 3 that Hb F contains two valyl residues in N-terminal position, while there are indica- ~ Dr. Huisman was unable to attend the Conference and this communication was presented by title but not read. It was announced at the time that it would be in- cluded in these Proceedings.

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DISCUSSION 251 tions that only one histidine and one tyrosine molecule are in C-terminal position.4 Chemical analyses of the total half-cystine content revealed the presence of six hal f-cystine residues per mole hemoglobin.5 The data ob- tained with two amperometric titration methods ~ Ingram6 and Benesch of alp) made it appear likely that fetal human hemoglobin contains four -SH groups. (Table I). The normal adult hemoglobin, on the contrary, contains TABLE I THE NUMBER OF SULFHYORYL GROUPS IN FETAL AND ADULT HUMAN HEMOGLOBIN ( M. W.68,000 ) Method Hb-A Hb F Assumed Assumed Chemical g.28 g 5.67 6 Amp. titrations;, ~ 7.6 8 4.0 4 Amp. titration ~ 9.6 4.4 Amp. titration + 4 M urea 7.6 8 4~1 4 fives 3 valyl residues in N-terminal position' one histidine and one tyrosine in C-terminal position) and eight half-cystine residues per mole hemoglobin, which are all present as free sulfhydryl groups.;; ~ (Table I>. The results obtained for fetal hemoglobin suggest that two half-cystine residues are present in ~ disulfide linkage in this protein. It therefore may be possible that either the Hb Fat is built up of two different polypeptide chains linked to each other by one disulEde bridge, or the disulfide bridge is present in one of the poly- peptide chains. In order to prove the first hypothesis some investigations were carried out by reducing this disulfide bridge with thioglycolic acid according to the method of Lindley.S With this technique it is possible, as shown for instance for insu- lin, to split a protein, which is built up by polypeptide chains linked by one or more -S-S- bridges, into the separate chains. The reduced protein was there- fore studied after coupling of the free -SH groups with iodoacetamide both by paper electrophoresis and by moving boundary electrophoresis. If after this reduction- two different polypeptide chains are formed, it may be possible to prove their existence by these electrophoretic techniques. The patterns for Hb F and Hb A are given in figure 1. The patterns for the fetal pigment ~ C, D and E ~ show two well-dis- tinguished components (marked 2 and 3 ~ while for the adult hemoglobin (A and B) one component was found (marked 1~. Electrophoretic studies of the two hemoglobins under the same conditions but without the reduction with thioglycolic acid (0.1 M glycine buffer solution pH S.1 with addition of 4 M urea) resulted in one component for both proteins. These results indicate that it is likely that fetal globin is divided into two different parts by the reduction with the thiol component. This supports the hypothesis that fetal hemoglobin i, built up by two different polypeptide chains connected by a disulfide bridge.

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PART III. ABNORMAL HEMOGLOBINS . ~ . ~ ~ ~ , .., FIG. 1. The electrophoretic pattern of the Hb A and Hb ~ after reduction with thioglycolic acid and coupling with iodoacetamide. Buffer solution: 0.1 M lithium thioglycolate pH 5.1 + 4 M urea (300 volts at 10 mA). A and Bnormal adult Hb; A after 2~/~ hours, B after 4~/~ hours. C, D and E fetal Hb; C after 3 hours, D after 44/~ hours, E after 5 hours. REFERENCES 1. Porter, R. R., and Sanger, F.: The free amino groups of haemoglobins, Biochem. J. 42: 287, 1948. 2. Schapira, G., and Dreyfus, J. C.: Groupes N-terminaux de l'hemoglobine de la maladie de Cooley, Compt. rend. Soc. de Biol. 148: 895, 1954. 3. Huisman, T. H. J., and Drinkwaard, J.: The N-terminal residues of five different human haemoglobins, Biochim. Biophys. Acta 18: 58S, 1955. 4. Huisman, T. H. J., and Dozy, A.: The action of carboxypeptidase on different human haemoglobins, Biochim. Biophys. Acta20: 400, 1956. 5. Hommes, F. A., Santema-Drinkwaard, J., and Huisman, T. H. J.: The sulihydryl groups of four different human haemoglobins, Biochim. Biophys. Acta 20: 564, 1956. 6. Ingram, V. M.: Sulphydryl groups in haemoglobins, Biochem. J. 59: 653, 1955. 7. Benesch, R. E., Lardy, H. A., and Benesch, R.: The sulfhydryl groups of crystal- line proteins. I. Some albumins, enzymes, and hemoglobins, J. Biol. Chem. 216: 663, 1955. S. Lindley, H.: The reduction of the disu]fide bonds of insulin, J. Am. Chem. Soc. 77: 4927, 1955.